Novel keratin-based dual-network hydrogel dressing as well as preparation method and application thereof

Abstract

The invention discloses a novel keratin-based dual-network hydrogel dressing as well as a preparation method and application thereof. The preparation method comprises the following steps: cross-linking chitosan, halloysite nanotubes and keratin to form a first network structure; tannic acid is introduced for further dynamic crosslinking with chitosan molecules, and a second network structure is formed; and freezing and drying to obtain the keratin-based double-network hydrogel dressing. According to the method, a freeze-drying technology is adopted, feather keratin (KP), halloysite nanotubes (HNTs) and chitosan (CS) are utilized, and the double-net-shaped hydrogel dressing which has rapid and effective hemostatic performance, antibacterial activity and biocompatibility and can promote healing is prepared. And a novel and efficient method is provided for wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of medical products, and in particular relates to a novel keratin-based double-network hydrogel dressing and a preparation method and application thereof. Background Art

[0002] The skin is an important barrier of the human body. Injuries to the skin can cause various symptoms, such as infection and inflammation, and can even be life-threatening in severe cases. The wound healing process is complex and involves hemostasis, inflammation, new tissue formation, and remodeling. Traditional wound dressings, such as gauze and bandages, mainly cover the wound to minimize infection, but do not actively promote the wound healing process. Instead, these dressings adhere to the wound surface by absorbing wound exudate, may cause pain when removed, and may damage new tissue. However, traditional wound dressings with a single function cannot meet the multiple needs of wound healing due to their limitations, so multifunctional wound dressings with healing effects are becoming the focus of research.

[0003] Bacterial infection is one of the main reasons for slow wound healing. Wound dressings with long-lasting antibacterial properties can usually significantly improve the healing effect, so the dressings need to have good antibacterial and adhesion properties. Hydrogels are widely used in the field of wound tissue recovery due to their good biocompatibility, adhesion and high water content. These properties can provide a moist environment and sufficient oxygen to promote cell proliferation.

[0004] Natural products can play an important role in wound healing. Due to their complex structure and biocompatibility, they can promote wound healing and prevent infection. It is imperative to use natural products to prepare multifunctional wound dressings with healing effects. Summary of the invention

[0005] This invention aims to significantly improve the antibacterial, hemostatic and healing effects of composite dressings by integrating hemostatic and antibacterial materials such as HNTs, CS and KP into them.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first object of the present invention is to provide a method for preparing a novel keratin-based double-network hydrogel dressing, which comprises the following steps: firstly cross-linking chitosan, halloysite nanotubes and keratin to form a first network structure; then introducing tannic acid to further dynamically cross-link with chitosan molecules to form a second network structure; and freeze-drying to obtain a keratin-based double-network hydrogel dressing.

[0008] Preferably, the mass ratio of chitosan, keratin, halloysite nanotubes and tannic acid is 1:(0.1-1.5):(0.1-1.5):(0.1-0.3).

[0009] Preferably, the mass ratio of chitosan, keratin, halloysite nanotubes and tannic acid is 1:(0.5-1):(0.5-1):0.2.

[0010] Preferably, the viscosity of the chitosan is 100-200 mPa.s.

[0011] Preferably, the method comprises the following steps:

[0012] 1) Dissolve CS in acetic acid aqueous solution and filter for later use;

[0013] 2) mixing the ultrasonicated HNTs with the above CS aqueous solution and stirring evenly;

[0014] 3) adding the regenerated keratin KP into the mixed solution of step 2) and stirring continuously;

[0015] 4) adding tannic acid to the mixed solution of step 3) and continuously stirring to obtain a keratin-based multifunctional composite hydrogel;

[0016] 5) freeze-drying the mixed solution in step 4) to obtain a keratin-based double-network hydrogel dressing.

[0017] Preferably, the specific steps are as follows:

[0018] 1) Weigh 2g CS and dissolve it in 2% by volume acetic acid aqueous solution, filter and set aside. The viscosity of CS is 100-200mPa.s;

[0019] 2) mixing the ultrasonicated HNTs with the above CS aqueous solution at a mass ratio of 1:1 and stirring for 1 hour;

[0020] 3) adding the regenerated keratin KP into the mixed solution of step 2) and stirring for 3 hours;

[0021] 4) adding tannic acid to the mixed solution of step 3) and continuously stirring for 8 hours to obtain a keratin-based multifunctional composite hydrogel dressing;

[0022] 5) placing the mixed solution in step 4) in a self-made mold for freezing, and then freeze-drying to obtain a keratin-based double-network hydrogel dressing.

[0023] The second object of the present invention is to provide a keratin-based double-network hydrogel dressing prepared by the above method.

[0024] The third object of the present invention is to provide the use of the above-mentioned keratin-based double-network hydrogel dressing in antibacterial, hemostatic and wound healing promotion.

[0025] Preferably, the wound surface is a wound surface infected by Staphylococcus aureus or Escherichia coli.

[0026] Preferably, the antibacterial agent is anti-Staphylococcus aureus or Escherichia coli.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) All hydrogel components are derived from nature, and each component is both a structural unit and a functional unit;

[0029] (2) The synthesis method is simple and mild, requiring only a self-crosslinking reaction at room temperature to ensure the biosafety and bioactivity of the material.

[0030] (3) The introduction of KP not only helps to quickly absorb blood in wounds with heavy bleeding, but also improves the antibacterial effect of the material and promotes wound healing.

[0031] (4) The introduction of HNTs not only improves the hemostatic rate of hydrogel dressings, but also regulates the pore structure of the hydrogel. With the increase of HNTs content, the hydrogel forms pore interconnected channels that are completely different from the traditional network structure, greatly improving the porosity of the material and effectively accelerating the absorption rate of wound exudate. The hemostatic rate of the new through-pore network structure is comparable to that of the layered structure.

[0032] (5) The present invention adopts a one-pot method to design and synthesize a keratin-based double-network multifunctional hydrogel, and combines the characteristics of natural products to enable the synthesized hydrogel to have multiple functions of adhesion, hemostasis, antibacterial and healing promotion. In vitro and in vivo experiments show that compared with pure CS / HNTs hydrogel materials, the hydrogel with the addition of keratin not only improves the liquid transfer rate, but also enhances the release of vascular endothelial growth factor through synergistic action with CS, promotes angiogenesis, and increases the deposition of collagen at the wound, thereby significantly accelerating wound healing in vivo. In the rat full-thickness wound model, the hydrogel group showed a significant wound healing rate of more than 95% on the 13th day, which was significantly better than the commercial control group. This new hydrogel provides a novel and efficient method for wound healing, is expected to play an important role in wound management, and has broad application prospects in the field of wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a physical picture of the keratin-based double network hydrogel of Example 3.

[0034] Figure 2 Schematic diagram of the double network hydrogel synthesis scheme of Example 3.

[0035] Figure 3 Actual pictures of keratin-based double-network aerogels of Examples 2 to 3.

[0036] Figure 4 These are SEM images of the keratin-based double network hydrogels of Examples 2 to 3 before and after hemostasis.

[0037] Figure 5 The antibacterial properties of the hydrogel materials of Control and Examples 1 to 3 against Staphylococcus aureus were tested.

[0038] Figure 6 The antibacterial properties of the hydrogel materials of Control and Examples 1 to 3 against Escherichia coli were tested.

[0039] Figure 7 The hemostatic properties of gauze and the hydrogel materials of Examples 1 to 3 on femoral artery injury in rats (n=6).

[0040] Figure 8 The figure shows the healing conditions of wounds infected with S. aureus without any dressing (M) and with the hydrogel material of Example 3.

[0041] Fig. 9 The figures show the healing conditions of wounds without any dressing (M) and wounds infected with the hydrogel material E.coil of Example 3. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below by specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial sources or by prior art methods unless otherwise specified. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0043] Embodiment 1:

[0044] (1) Weigh 2 g of CS at a mass volume ratio of 2% and dissolve it in 100 mL of acetic acid aqueous solution (the volume ratio of acetic acid to water is 2:100), filter and set aside. The viscosity of CS is 100-200 mPa.s.

[0045] (2) HNTs (500 mg) after 40 kHz ultrasound for 30 min were mixed with the above CS aqueous solution (25 mL) (the mass ratio of HNTs to CS was 1:1) at 900 rpm and stirred for 1 hour.

[0046] (3) Add 100 mg of tannic acid to the mixed solution of step (2) and continue stirring (900 rpm) for 8 hours to obtain chitosan hydrogel.

[0047] (4) The above-mentioned CS was placed in a homemade mold and frozen, and CS aerogel dressing (CS-1HCNs-SM) was obtained by freeze-drying method.

[0048] (5) The antibacterial activity of the hydrogel was tested using S. aureus and E. coil as test strains. The bacteria were reactivated twice for the experiment, and the bacterial stock solution was added to the sterilized LB liquid culture medium and cultured for 12 h at 37 °C and 150 rpm / min. Before bacterial culture, the gel sample was exposed to ultraviolet light for 24 h. The initial concentration of the bacterial suspension was adjusted to 10 7 CFU / mL, 10 mg of the hydrogel prepared in step (3) (without adding hydrogel as the control) was mixed with 25 mL of the bacterial solution and placed in a test tube, and placed on a constant temperature incubator shaker at 37 ° C and 120 r / min for 1 h. Then, 100 μL of the suspension was inoculated on a sterilized LB agar plate, and the number of bacteria was counted after incubation in a 37 ° C incubator for 24 h, and the inhibition rate was calculated.

[0049] (6) The ethical requirements for all animals in the animal experiments in this work strictly followed the National Research Council's Guide for the Care and Use of Laboratory Animals. Male SD rats weighing between 400 and 500 g were selected as the femoral artery injury model because of their less bleeding. Before surgery, sodium pentobarbital anesthesia was usually used, and the rats were fixed on the operating board to ensure the accuracy of the operation and the stability of the animals. First, the hair in the groin of the rat's thigh was shaved to expose the skin, which was then disinfected with 75% ethanol solution, and then an incision was made to expose the femoral artery. Subsequently, the femoral blood vessels were cut, resulting in heavy bleeding. Immediately, pre-sterilized aerogel (diameter: 3 cm) was applied to the wound. The coagulation was observed every 10 seconds until the bleeding stopped completely. The coagulation time was recorded, and the blood loss was calculated after weighing the hemostatic material. The wound was photographically recorded. Three rats were used in each group.

[0050] (7) The mice were anesthetized with 10% ether, depilated, and disinfected with 75% alcohol. Six groups of circular wounds with a diameter of about 8 mm were made on the back of the mice using a hole puncher. 50 μL of 1.0×10 7 CFU / mL of Staphylococcus aureus or Escherichia coli liquid was infiltrated for 1 hour to construct two infectious wound models. The wound surface without any dressing was set as the blank control group, and the hydrogel dressing prepared in step (3) was fixed on the wound surface as the experimental group, and the dressing was replaced once every 3 days. The wound area was recorded on the 0th, 3rd, 6th, 10th and 14th day.

[0051] Embodiment 2:

[0052] (1) Same as Example 1-(1).

[0053] (2) The HNTs after ultrasonic treatment (40 KHz, 30 min) were mixed with the above CS aqueous solution (the mass ratio of HNTs to CS was 0.5:1, 250 mg and 500 mg, respectively) and stirred (900 rpm) for 1 hour.

[0054] (3) Add 500 mg of regenerated keratin KP to the mixed solution of step (2) and continue stirring (900 rpm) for 3 hours.

[0055] (4) Add 100 mg of tannic acid to the mixed solution of step (3) and continue stirring for 8 hours to obtain a keratin-based multifunctional composite hydrogel dressing (N-CSKP-0.5HCNs-SM).

[0056] (5) The mixed solution in step (4) is placed in a self-made mold for freezing, and then freeze-dried to obtain a keratin-based multifunctional composite aerogel. Figure 3 As shown, the SEM images before and after hemostasis are as follows Figure 4 shown.

[0057] (6) Similar to Example 1-(5), the hydrogel with added keratin has more efficient antibacterial activity.

[0058] (7) Same as Example 1-(6).

[0059] (8) Similar to Example 1-(7), the hydrogel with added keratin can shorten the inflammatory phase of wound tissue and allow the wound surface to enter the cell proliferation phase more quickly, thereby achieving antibacterial debridement and accelerating wound healing.

[0060] Embodiment 3:

[0061] (1) Same as Example 1-(1).

[0062] (2) 500 mg of HNTs after ultrasound (40 KHz, 30 min) were mixed with the above CS aqueous solution (25 mL) (mass ratio of 1:1) and stirred for 1 hour at a rotation speed of 900 rpm.

[0063] (3) Add 500 mg of regenerated keratin KP to the mixture in step 2 and continue stirring for 3 hours at a speed of 900 rpm.

[0064] (4) Add 100 mg of tannic acid to the mixed solution in step 3 and continue stirring for 8 hours to obtain a keratin-based multifunctional composite hydrogel dressing (N-CSKP-1HCNs-SM). Figure 1 As shown in Figure 1, the hydrogel forms a double network structure that is completely different from the traditional network structure, with different pores interconnecting channels. The schematic diagram of the synthesis scheme of the double network hydrogel is shown in Figure 1. Figure 2 shown.

[0065] (5) The mixed solution in step 4 is placed in a homemade mold for freezing, and then freeze-dried to obtain a keratin-based multifunctional composite aerogel. Figure 3 As shown in the SEM images before and after hemostasis, Figure 4 shown.

[0066] (6) Similar to Example 1-(5), the hydrogel with added keratin has more efficient antibacterial activity.

[0067] (7) Same as Example 1-(6).

[0068] (8) As in Example 1-(7), the healing effect of N-CSKP-1HCNs-SM on wounds infected with S. aureus was as follows: Figure 8 As shown in Figure 2, the healing of E.coil infected wounds is as follows: Fig. 9 As shown. In the rat full-thickness wound model, the hydrogel group showed a significant wound healing rate of over 95% on the 13th day, which was significantly better than the commercial control group. The hydrogel with added keratin can shorten the inflammatory phase of wound tissue and allow the wound surface to enter the cell proliferation phase faster, achieving antibacterial debridement and accelerating wound healing.

[0069] Test example:

[0070] 1. Calculation of bacterial inhibition rate:

[0071] (1) Calculation formula:

[0072] Inhibition rate (%) = (bacterial number of blank group - bacterial number of sample group) / bacterial number of sample group.

[0073] (2) Results:

[0074] The antibacterial properties of the hydrogel materials of Examples 1 to 3 against Staphylococcus aureus are as follows Figure 5 The antibacterial properties against Escherichia coli are shown in Figure 6 shown.

[0075] 2. Hemostasis performance test:

[0076] The hemostatic properties of the hydrogel materials in Examples 1 to 3 on femoral artery injury in rats are as follows Figure 7 shown.

[0077] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing a novel keratin-based double-network hydrogel dressing, characterized in that: The method comprises the following steps: firstly cross-linking chitosan, halloysite nanotubes and keratin to form a first network structure; then introducing tannic acid to further dynamically cross-link with chitosan molecules to form a second network structure; and freeze-drying to obtain a keratin-based double-network hydrogel dressing.

2. The method according to claim 1, characterized in that The mass ratio of chitosan, keratin, halloysite nanotubes and tannic acid is 1:(0.1-1.5):(0.1-1.5):(0.1-0.3).

3. The method according to claim 2, characterized in that The mass ratio of chitosan, keratin, halloysite nanotubes and tannic acid is 1:(0.5-1):(0.5-1):0.

2.

4. The method according to claim 1, characterized in that: The viscosity of the chitosan is 100-200 mPa.s.

5. The method according to claim 1, characterized in that The steps include: 1) Dissolve CS in acetic acid aqueous solution and filter for later use; 2) mixing the ultrasonicated HNTs with the above CS aqueous solution and stirring evenly; 3) adding the regenerated keratin KP into the mixed solution of step 2) and stirring continuously; 4) adding tannic acid to the mixed solution of step 3) and continuously stirring to obtain a keratin-based multifunctional composite hydrogel; 5) freeze-drying the mixed solution in step 4) to obtain a keratin-based double-network hydrogel dressing.

6. The method according to claim 5, characterized in that The specific steps are as follows: 1) Weigh 2g CS and dissolve it in 2% by volume acetic acid aqueous solution, filter and set aside. The viscosity of CS is 100-200mPa.s; 2) mixing the ultrasonicated HNTs with the above CS aqueous solution at a mass ratio of 1:1 and stirring for 1 hour; 3) adding the regenerated keratin KP into the mixed solution of step 2) and stirring for 3 hours; 4) adding tannic acid to the mixed solution of step 3) and continuously stirring for 8 hours to obtain a keratin-based multifunctional composite hydrogel dressing; 5) placing the mixed solution in step 4) in a self-made mold for freezing, and then freeze-drying to obtain a keratin-based double-network hydrogel dressing.

7. A keratin-based double-network hydrogel dressing prepared by the method according to any one of claims 1 to 6.

8. Use of the keratin-based double-network hydrogel dressing according to claim 7 in antibacterial, hemostatic and wound healing.

9. The use according to claim 8, characterized in that: The wound surface is a wound surface infected by Staphylococcus aureus or Escherichia coli.

10. The use according to claim 8, characterized in that: The antibacterial agent is anti-Staphylococcus aureus or Escherichia coli.

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